Retroviral vectors for gene therapy

ABSTRACT

The present invention provides novel, improved retroviral vectors which can be used for gene therapy, more specifically, retroviral vectors which are not only safer, more versatile, and more convenient than any other existing vectors, but they also drive high levels of gene expression and high viral titer. In retroviral vectors of the present invention, gag and env coding sequences are deleted, and all or part of U3 can be readily substituted with heterologous, non-retroviral promoter elements. Furthermore, at least one internal ribosome entry site is employed to express more than one genes, and multicloning sites are placed in an insertion site for cloning of a heterologous promoter or a foreign gene.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel, improved retroviral vectorswhich can be used for gene therapy, more specifically, to retroviralvectors which are not only safer, more versatile, and more convenientthan any other existing vectors, but they also drive high levels of geneexpression and high viral titer.

2. Description of the Prior Art

Murine leukemia virus(MLV)-based retroviral vectors which are the mostwidely used gene delivery vehicles in gene therapy clinical trials, havebeen employed in almost 70% of approved protocols(see: Ali, M. et al.,Gene Ther., 1:367-384, 1994; Marshall, E., Science, 269:1050-1055,1995).

However, despite their frequent use for gene transfer, many of thebiochemical and genetic properties of MLV such as cis and trans factorsimportant for gene expression, viral assembly and packing, have not beencompletely understood. Moreover, there are many problems with theretroviral vectors currently in clinical use such as MFG(see: Bowtell,D. et al., J. Virol., 62:2464-2473, 1988; Ohashi, T. S. et al., Proc.Natl. Acad. Sci., USA, 89:11332-11336, 1992; Jaffe, E. M., Cancer Res.,53:2221-2226, 1993; Bender, M. A. et al., J. Virol., 61:1639-1646,1987), and LN-based vectors (see: Boggs, S. S. et al., Gene Ther.,2:632-638,1995; Armentano, D. et al., J. Virol., 61:1647-1650, 1987;Adam M. A., and A. D. Miller, J. Virol., 62:3802-3806, 19B8; Osborne, W.R. A., and A. D. Miller, Proc. Natl. Acad. Sci., USA, 85:6851-6855,1988; Miller, A. D., and Roseman, Biotechniques, 7:980-990, 1989;Palmer, T. D. et al., Blood, 73:438-445, 1989).

First, all retroviral vectors contain sequences which are also presentin the packaging lines. Accordingly, recombination between the packaginggenome and the vector results in the generation of replication-competentretrovirus(RCR).

Secondly, most retroviral vectors use either long terminal repeat(LTR)sequences from MLV or related LTR sequences such as myeloidproliferation stimulating virus(MPSV), murine sarcoma virus(MSV) or anheterologous internal promoter. Although the LTR works efficiently incertain cell types, its activity can be down-regulated and its presencecan affect expression from internal promoters(see: Bowtell, D. D. L. etal., J. Virol., 62:2464-2473, 1988; Emerman, M. and H. M. Temin, Cell,39:449-467, 1984).

Thirdly, the viral titers achieved with the vectors in current packaginglines, although they are improved ones, are still not sufficiently highenough for many therapeutic applications.

Fourthly, MLV-based vectors packaged in murine packaging lines, aresusceptible to complement-mediated inactivation in vivo, which, in turn,limits their utility for in vivo applications(see: Takeuchi, Y. et al.,J. Virol., 68:8001-8007, 1994; Cosset F. L. et al., J. Virol.,69:7430-7436, 1995).

Fifthly, it is difficult to produce a virus at a reasonable titer fortargeting a specific cell type or tissue by direct, in vivodelivery(see: Kasahara, N. et al., Science, 266:1373-1376, 1994; KabatD., Science, 269:417, 1995).

Finally, MLV-based vectors, when packaged in murine packaging lines,cannot deliver a gene of interest to non-dividing cells.

Under the circumstances, WO 92/07943 discloses a retroviral vector MFGincluding an insertion site for genes of interest which are capable ofexpressing high levels of the protein derived from the genes of interestin a wide variety of transfected cell types, and those lacking aselective marker, thus rendering them suitable for human gene therapy inthe treatment of a variety of disease states without the co-expresssingof a selective marker.

In this connection, the present inventors compared the levels of geneexpression from several types of retroviral vectors currently used inclinical trials for gene therapy(see: Byun, J. et al., Gene Ther.,3:780-788, 1996). As a result, it has been suggested that the MFGretroviral vector is superior in conferring gene expression aftertransduction of a variety of target cells, whose results are consistentwith the previous reports in the art(see: Ohashi, T. S. et al., Proc.Natl. Acad. Sci., USA, 89:11332-11336, 1992; Riviere I. et al., Proc.Natl. Acad. Sci., USA, 92:6733-6737, 1995; Krall, W. J. et al., GeneTherapy, 3:37-48, 1996).

However, it has been found that the MFG retroviral vector has manyfeatures that should be modified in terms of gene expression, viraltiters and safety as follows:

First, MFG contains significantly long coding sequences for gag and envused as a template for homologous recombination, which increase thepossibility of the generation of replication-competent retrovirus (RCR).

Secondly, MFG-mediated gene expression is driven by the MLV LTR which isa medium-strength constitutive promoter. However, for many therapeuticapplications, it would be necessary to regulate gene expression, notonly in terms of levels but also timing of expression, in asophisticated manner. Accordingly, if heterologous promoter elements areinserted to U3 or U3 is replaced with other full-size promoters, theretroviral vector can be applied in a wide variety of gene therapy. Inthis connection, the enhancer region of MLV U3 is replaced withheterologous enhancers and U3 is substituted with U3 from similarretroviruses such as MPSV and Friend MLV, nevertheless, it is not clearwhether U3 can be replaced with a completely different sequence.

Thirdly, the original version of MFG is designed for expression of asingle gene, though the expression of more than two or three genes isdesirable for many therapeutic applications.

Finally, NcoI which coincides with the ATG codon of the env gene shouldbe the expression site in MFG to obtain high levels of gene expression,which, in turn, restricts the broader use of MFG retroviral vector.

Naturally, in order for retroviral vectors including MFG to beclinically viable forms of gene delivery, some or all of the currentlimitations have to be addressed. Accordingly, there are strong reasonsfor exploring novel and improved retroviral vectors which are capable ofexpressing controlled levels of proteins derived from the genes ofinterest in a wide range of transfected cells.

SUMMARY OF THE INVENTION

In accordance with the present invention, the inventors discovered thatseveral retroviral vectors in which gag and env sequences unnecessaryfor packaging are deleted, all or part of U3 sequence inessential forretroviral functions is replaced with heterologous promoter elements, atleast one internal ribosome entry site is employed to express more thanone genes, and multicloning sites are placed in an insertion site forcloning of a heterologous promoter or a foreign gene, can drive highlevels of gene expression and high viral titer.

A primary object of the invention is, therefore, to provide novel,improved retroviral vectors for gene therapy, in terms of safety,versatility and convenience, which can drive high levels of geneexpression and high viral titer.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and the other objects and features of the present inventionwill become apparent from the following descriptions given inconjunction with the accompanying drawings, in which:

FIG. 1 is a schematic representation of a retroviral vector MFG.

FIG. 2 is a schematic diagram for the construction of vectors containingdeletions at the region around the packaging signal sequence.

FIG. 3 is a schematic representation of the vectors containing deletionsof FIG. 2.

FIG. 4A is a histogram showing effects of deletion on gene expression.

FIG. 4B is a histogram showing effects of deletion on viral titers.

FIG. 5 is a schematic diagram for the construction of a vector in whicha residual env coding sequence is deleted.

FIG. 6A is a schematic representation of the vector in which theresidual env coding sequence is deleted.

FIG. 6B is a histogram showing CAT(chloramphenicol acetyltransferase)activity of CRIP packaging cells transfected with MΔE-CAT plasmid.

FIG. 6C is a histogram showing CAT activity of human promonocytic cellline U937 transducted with cell-free viral supernatants obtained fromthe transfected packaging cells of FIG. 6B.

FIG. 6D is a histogram showing CAT activity of T lymphoid cell line CEMtransducted with cell-free viral supernatants obtained from thetransfected packaging cells of FIG. 6B.

FIG. 7 is a schematic representation on four fragments from the majorimmediate-early promoter of human cytomegalovirus, which are to be usedto substitue the U3 sequence.

FIG. 8 is a schematic diagram for the construction of vectors containingfour fragments of FIG. 7.

FIG. 9 is a schematic diagram for the construction of a cloning vectorRPX68.

FIG. 10 is a schematic diagram of chimeric LTRs.

FIG. 11 is a schematic diagram for the construction of retroviralvectors containing hybrid U3 at the 5′LTR.

FIG. 12 is a schematic diagram for the construction of retroviralvectors containing hybrid U3 at the 3′LTR.

FIG. 13A is a schematic representation of 4 retroviral vectorscontaining hybrid U3 at the 3′LTR.

FIG. 13B is a histogram showing CAT activity of CRIP packaging cellstransfected with retroviral vectors of FIG. 13A.

FIG. 13C is a histogram showing CAT activity of cell line NIH3T3transducted with cell-free viral supernatants obtained from thetransfected packaging cells of FIG. 13B.

FIG. 13D is a histogram showing CAT activity of H9 cells transductedwith cell-free viral supernatants obtained from the transfectedpackaging cells of FIG. 13B.

FIG. 14 is a schematic diagram for the construction of a retroviralvector containing hybrid U3 at both 5′ and 3′LTRs.

FIG. 15 is a schematic diagram for the construction of CAT-expressingretroviral vectors without gag and env coding sequences, but containingthe hybrid U3 and IRES.

FIG. 16 is a schematic diagram for the construction of the IRES-NEOcassette.

FIG. 17 is a schematic representation of several retroviral vectorscontaining IRES and NEO, which are prepared by the construction schemeof FIG. 15

FIG. 18A is a schematic representation of retroviral vectors MFG-mGM/CSFand SCP1-mGM/CSF containing a murine GM-CSF gene as a heterologous gene.

FIG. 18B is a histogram showing expression levels of mGM-CSF from CRIPpackaging cells transfected with retroviral vectors of FIG. 18A.

FIG. 18C is a histogram showing expression levels of mGM-CSF from HFFcells transducted with cell-free viral supernatants obtained from thetransfected packaging cells of FIG. 18B.

FIG. 18D is a histogram showing expression levels of mGM-CSF from NIH3T3cells transducted with cell-free viral supernatants obtained from thetransfected packaging cells of FIG. 18B.

FIG. 19A is a schematic representation of retroviral vectors MFG-WIN,KCP3-WNIN, and KCP3-WXIN.

FIG. 19B is a histogram showing effect of gene expression of theretroviral vectors of FIG. 19A.

FIG. 20 is a schematic diagram for the construction of improvedretroviral vectors COI, CTI, and COE.

FIG. 21 is a schematic diagram for the construction of improvedretroviral vectors MOI, MTI, and MOE.

FIG. 22A is a schematic diagram for the construction of improvedretroviral vector MOMT12.

FIG. 22B is a schematic diagram for the construction of retroviralvector MOMT13.

FIG. 22C is a schematic representation of retroviral vectors MOMT12 andMOMT13.

FIG. 23A is a schematic representation of retroviral vectors MOI,MOIN-EPO, COI, and COIN-EPO.

FIG. 23B is a histogram showing effect of gene expression of theretroviral vectors MFG-WIN, MOIN-EPO, and COIN-EPO, together withrelative virus titers shown at the bottom of the histogram.

FIG. 24 is a schematic representation of 5′LTR, EPO and 3′LTR positionsto be amplified by PCR on retroviral vectors MFG-WIN, MOIN-EPO, andCOIN-EPO.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have constructed several novel and improvedretroviral vectors that can be used for gene therapy, by employing aMLV-based retroviral vector, preferably a retroviral vector MFG(ATCC68754), as a starting vector for systematic deletion analysis andmodification. The retroviral vectors of the present inventionaccommodate the following features:

1. They contain the minimum length of retroviral sequences in which gagand env coding sequences which do not significantly affect viralfunctions such as packaging or gene expression are deleted. Inparticular, since gag coding sequence and its immediate upstream regionare included in all currently available retroviral vector, and aretroviral vector like MFG contains a significantly long env codingsequence, deletions of gag and env sequences decrease the possibility ofhomologous recombination between the nucleotide sequences in the vectorand the packaging line and of generation of RCR.

2. All or part of U3 sequence in the 3′LTR is substituted with full-sizeheterologous promoters or their fragments with any deleterious effects,therefore making it possible to control gene expression in a precisemanner.

3. They contain restriction sites at the truncated U3 of the 3′LTR, sothat heterologous promoter elements can be easily inserted thereto. Thisway, retroviral vectors can be readily converted for different purposes,for example, by altering levels and regulatory modes of gene expression.

4. They contain one or two IRESs so that two or three genes can beexpressed in one vector as a multicistronic message.

5. They contain multicloning sites at the expression site, which easesthe convenient insertion of foreign genes.

The present retroviral vectors having the aforementioned characteristicscover retroviral vectors which are capable of delivering a gene ofinterest to a target cell when packaging functions of Gag, Pol and Envare provided, and are free of gag coding sequence. The retroviralvectors are based on MLV in which nucleotide sequence of #523 to #1040including entire gag coding sequence is deleted.

From the retroviral vectors, entire env coding sequence, and retroviralnuclotide sequence between termination codon of env coding sequence andpolypurine tract can be further removed. In addition, in the retroviralvectors, the gene of interest is located downstream from both a splicedonor site and a splice acceptor site.

The gene of interest that is incorporated in the vectors of theinvention may be any gene which produces a hormone, an enzyme, areceptor or a drug(s) of interest.

The retroviral vectors of the invention are further characterized inthat all or part of at least one U3 sequence of a 5′LTR and a 3′LTRderived from a retrovirus is substituted with a full-size heterologouspromoter or a fragment thereof. At this time, the full-size heterologouspromoter or the fragment thereof is inserted at the truncated U3 ofMLV-based retroviral vector in which truncation is provided at thenucleotide sequence which is selected from the group consisting of: anucleotide sequence between #−330 Pvu II and #−152 Xba I recognitionsites on the 5′LTR, a nucleotide sequence between #−152 Xba I and #−36Sac I recognition sites on the 5′LTR, a nucleotide sequence between#−330 Pvu II and #−36 Sac I recognition sites on the 5′LTR, a nucleotidesequence between #−419 Nhe I and #−36 Sac I recognition sites on the5′LTR, and nucleotide sequences on the 3′LTR corresponding to saidnucleotide sequences on the 5′LTR.

In describing the retroviral vector of the present invention, thenumbering is carried out, based on Shinnick et al's publication(see:Shinnick et al, Nature, 293:543-548, 1981).

In the retroviral vectors of the invention, the heterologous promoter isregulated at the transcription level by the aid of a chemical or abiological molecule, where the biological molecule may be any materialsof hormone, growth factor, enzyme, lymphokine or cytokine.

Moreover, the retroviral vectors may further comprise one or twointernal ribosomal entry site(IRES) which are obtainable from murineencephalomyocarditis virus (EMCV) or foot and mouth disease virus(FMDV).It is preferred that each of the two IRES is obtained from differentviral sources, in case of employing two IRES.

The retroviral vectors may further comprise a multicloning site at thetruncated U3 of the 3′LTR, so that the full-size heterologous promoteror the fragment thereof can be easily inserted thereto, or amulticloning site downstream from the splice acceptor site or upstreamfrom the IRES, so that a gene of interest can be easily insertedthereto.

The present invention is further illustrated in the following examples,which should not be taken to limit the scope of the invention.

Cell lines, culture media therefor and a method for the determination oftransfection efficiency and viral titer which are employed in Examplesbelow, are as follows:

CRIP(CRL 9808), NIH3T3(CRL1658) and U937(CRL1593) were obtained from theAmerican Type Culture Collection(ATCC) (Rockville, Md., U.S.A.), whileCEM-SS(#776 or #87) was from the NIH AIDS Research and Reference ReagentProgram(Rockville, Md., U.S.A.). BING is an amphotropic cell linederived from 293T cell(see: DuBridge, R. B. et al., Mol. Cell. Biol.,7:379-387, 1987) which is similar to the ecotropic BOSC 23 packagingcell line(see: Pear, W. S. et al., Proc. Natl. Acad. Sci., USA,90:8392-8396, 1993).

NIH3T3 and CRIP were grown in Dulbecco's modified Eagle's medium(D-MEM)supplemented with 10% fetal bovine serum(FBS). CEM-SS, H9 and U937 weregrown in RPMI 1640 medium supplemented with 10% FBS. Each medium used inthe Examples was supplemented with 120 μg/ml penicillin G(Sigma P-3032;1690 U/mg) and 200 μg/ml streptomycin sulfate(Sigma S-9137; 750 U/mg).

BING and CRIP were transfected by a calcium phosphate-DNAcoprecipitation method as previously described in detail(see: Byun, J.et al., Gene Ther., 3:780-788, 1996; Pear, W. S. et al., Proc. Natl.Acad. Sci., USA, 90:8392-8396, 1993; Miller, A. D. and C. Buttimore.,Mol. Cell. Biol., 6:2895-2902, 1986). A total of 10 μl DNA in 500 μlCaCl₂/H₂O (124 mM CaCl₂) was mixed with 500 μl of 2×HBS(280 mM NaCl, 10mM KCl, 1.5 mM Na₂HPO₄.2H₂O, 12 mM dextrose, 5 mM HEPES) with constantbubbling and within 1 to 2 minutes, this solution was added to the cellswith 25 mg/ml of chloroquine. Transfection efficiency was measured inmost experiments by X-gal(5-bromo-4-chloro-3-indolyl-β-γ-galactopyranoside) staining using thesame culture plates or duplicate dishes, if necessary.

Supernatants from the transfected packaging cells were collected,usually 48 hrs after transfection, filtered through a 0.45 μm filter,and used for transduction of target cells. For transduction of CEM-SS,H9 and U937, 5×10⁶ cells were harvested, resuspended with 5 ml viralsupernatant in the presence of 8 μg/ml polybrene, and incubated in a 37°C. incubator (5% CO₂) with occasional stirring for 5 hrs. Fresh mediumwas then added to maintain the cell density at 5 to 6×10⁵ cells/ml andgrown for another 36 to 44 hrs. NIH3T3 was also transduced with 3 ml ofviral supernatant in the presence of 8 mg/ml polybrene for 5 hrsfollowed by the addition of fresh medium. On the following day, thecells were re-fed with fresh medium containing G481 as required. Whenneeded, viral titer was determined in accordance with the Byun et al'smethod(see: Byun, J. et al., Gene Ther., 3:1018-1020, 1996).

EXAMPLE 1 Defining the Packaging Sequence in MFG

A schematic diagram depicting a retroviral vector MFG(ATCC 68754) isprovided in FIG. 1. In MFG, a gene of interest(dotted box) is clonedinto the Nco I site containing the start codon, and expressed as aspliced message. MFG contains the 420 bp and 99 bp coding sequences forgag and env, respectively. U3 of MoMLV(Molony murine leukemia virus) is448 bp long. In FIG. 1, ATG is a start codon of gag, SD is a splicedonor site, and SA is a splice acceptor site.

MFG contains gag sequence up to the Nar I site at position #1040followed by a splice acceptor fragment from the Nde 1 site(#5402) to theXba l site(#5766) in MLV(see: FIG. 1). An adapter oligonucleotide wasused to insert an NcoI site at position 7675, converted to a BamH Isite, to the end of MLV. The gene inserted at the NcoI site is expressedfrom a spliced message, resembling the normal spliced env messagefollowing MLV infection.

Initially the present inventors were interested in determining whetherthe gag and env regions of the vector were essentially required forviral functions or not. The former is thought to contain the sequencenecessary for viral packaging, whereas the latter does not seem to beneeded for any retroviral vector functions. These sequences will enhancethe frequency of recombination between the packaging genome and thevector, increasing the possibility of producing RCR. Furthermore,deletion of unnecessary sequences will allow the insertion of larger DNAfragments into the vector.

To determine the minimum length of nucleotide sequence required forpackaging, a series of deletions between the splice donor and acceptorwere generated by polymerase chain reaction(PCR) using proof-reading pfuDNA polymerase(Stratagene, USA) (see: FIG. 2). The nucleotide sequencesof final constructs were determined to confirm that there were nomutations introduced by this amplification step.

As can bee seen in FIG. 2, based on MFG-LacZ(see: Bandara, G. et al.,Proc. Natl. Acad. Sci., USA, 90:10764-10768, 1993), ten oligonucleotideprimers were used for amplification of two types of fragment, Groups Iand II. Group I fragments were obtained by PCR using the primer HindIIIR and one of the four primers L228, L377, L523, and L739. The HindIIIlinker was attached to Hind IIIR, while the Xho I linker was attached tothese L series primers. Group II fragments were generated by PCR usingthe primer ClaIL and one of the four primers R371, R527, R743, andR1016. The Cla I and Xho I linkers were attached to respective primers.The nuclotide sequence of primers used in this experiment is as follows:

HindIIIR SEQUENCE ID NO:1:     GCATTAAAGCTTTGCTCT          Hind III L228SEQUENCE ID NO:2: GCCTCGAGATAAGTTGCTGGCCAG    Xho I L377 SEQUENCE IDNO:3: GCCTCGAGTCCCTGGGACGTCTCC    Xho I L523 SEQUENCE ID NO:4:GCCTCGAGCAAAAATTCAGACGGA    Xho I L739 SEQUENCE ID NO:5:GCCTCGAGCAGAAGGTAACCCAA    Xho I R371 SEQUENCE ID NO:6:GCCTCGAGGGACTTCGGGGGGCCGT    Xho I R527 SEQUENCE ID NO:7:GCCTCGAGGTTTGGGACCGAAGCC    Xho I R743 SEQUENCE ID NO:8:GCCTCGAGAATGGCCAACCTTTAA    Xho I R1016 SEQUENCE ID NO:9:GCCTCGAGCCCTCACTCCTTCTCT    Xho I ClaIL SEQUENCE ID NO:10:ACGCTCATCGATAATTTC        Cla I

The 8 fragments from Group I and II were amplified and cloned into theplasmid pCR II(Invitrogen, USA), resulting in a series of 4 pCR II-I and4 pCR II—II constructs. The Xho I-Xho I fragments were isolated from theseries of pCR II—II plasmids, and then inserted into the Xho I site ofthe series of pCR II-I, generating a series of pMAgag constructs. TheHind III-Cla I fragment was isolated from pMAgag and used to replace theHind III-Cla I fragment(including the 5′LTR) of MFG-LacZ, resulting in aseries of pMΔLacZ constructs, now containing deletions between SD andSA. Altogether 9 deletion mutants were constructed, as summarized inFIG. 3, and their effects tested on packaging and transductionefficiencies, using the lacZ gene as a reporter.

In FIG. 3, ψ indicates the packaging sequence previously defined by Mannet al.(see: Mann et al., Cell, 33:153-159, 1983) which includes the gagcoding region as well as the entire sequence between SD and the startcodon for gag. The numbering system is based on Shinnick et al'spublication(see: Shinnick et al, Nature, 293:543-548, 1981). The regionbetween 1040 and 5400 includes coding sequences for gag and pol and ismissing from MFG. LacZ was used as a reporter gene in this study and itsrelative position is shown as dotted triangle.

Deletion constructs, together with the parental vector MFG-lacZ, weretransfected to the packaging line BING. After 3 days, culturesupernatants were filtered through 0.45 μm filters, while cells werestained with X-gal to measure transfection efficiency. Duplicate disheswere also prepared for some constructs and subjected too-nitrophenyl-β-D-galactopyranoside(ONPG) assay for β-galactosidaseactivity. X-gal-stained cells were counted to estimate the packagingefficiency(see: FIG. 4A).

Cell-free viral supernatants were used to transduce NIH3T3 cells andafter 3 days, cells were stained with X-gal to determine viral titer.Transfection efficiency was determined by measuring both lacZ activityand the number of X-gal stained cells in the transfected packaging line.All mutant constructs gave comparable numbers of blue cells withvirtually identical intensity as well as similar levels of lacZactivity, demonstrating that the deletions did not affect geneexpression(see: FIG. 43).

Transfection and transduction efficiency of MFG were set to 1 and thoseof others were normalized to it. More than 5 transfection andtransduction experiments were performed at separate times. In oneindependent experiment, 4 to 6 transfection and transduction experimentswere carried for each mutant.

The relative titers of each of the deletion constructs is shown in FIG.4B. The deletion constructs can be divided into three classes, dependingon their effects on packaging efficiency. First, four mutant constructscontaining the deletion from 228 to 371 (Δ15, Δ16, Δ17, Δ18) completelylost the packaging function. Secondly, the sequence from 377 to 527appeared to be necessary, but not essential, for the optimal packagingefficiency since the titers of these mutant constructs (Δ26, Δ27, Δ28)were consistently lower than control. Thirdly, there are two mutantconstructs that reproducibly showed a maximally 2-fold increase inpackaging efficiency. The mutant construct Δ38, which always gave thehighest titer, contains a 500 bp deletion removing the entire gag codingsequence present in MFG.

The mutational analysis of the packaging signal produced the three typesof phenotype(no packaging, decreased packaging, and increased packaging)and defined at least 3 regions involved in packaging. The first group ofmutants, all of which contained a deletion in Region A (#228 to #371)showed absolutely no packaging function, is consistent with previousreports(see: Mann, R. et al., Cell, 33:153-159, 1983; Mann, R., and D.Baltimore., J. Virol., 54:401-407, 1985; Alford, R. L. et al., Virology,183:611-619, 1991). The second phenotype is characterized by 2 folddecrease in packaging, localizing the region that are not essential butnecessary for the maximum packaging function. The third group ofdeletion mutants, Δ38 and Δ48, reproducibly showed maximally 2-foldhigher packaging efficiency than the parental type, suggesting thepossible presence of the sequence interfering with the packagingfunction, probably at the 739 to 1016 region (Region C). In mutant Δ38which contains almost a 500 bp deletion, the entire gag coding sequencewas removed, but showed no decrease in packaging efficiency. However,when deletion is extended to 377(Region B) as in Δ28, packagingefficiency was decreased substantially.

In summary, there seems to be a complex array of sequences that areinvolved in viral packaging; Region A essential for viral packaging,Regions B which is necessary, but not essential, for optimal packaging,and Region C which probably interferes with packaging. When both RegionsB and C were deleted, the B phenotype was shown. These results show thatthe entire N-terminal gag sequence is not necessary for efficient viralpackaging in the context of the MFG vector. The retroviral vectorlacking the gag coding region produced viral titer and levels of geneexpression similar to the one containing this sequence even when variousreporter genes including CAT, EPO and GM-CSF were used, as shown inExamples later.

EXAMPLE 2 Deletion of the Residual Env Sequence

MFG also has approximately 140 bp between the stop codon of the foreigngene and the 5′end of U3(see: FIG. 1 and FIG. 6A). This region containsthe 99 bp env coding sequence which can be used as a template forrecombination with the same sequence in the packaging line. We deleted113 bp, including the entire residual env coding sequence, but left thepolypurine tract intact(see: FIG. 6A).

To remove the residual env sequences, the PCR was performed withMFG-NEO(see: Byun, et al., Gene Ther., 3:780-788, 1996) using theprimers M3L-52 and M3L-3l, as illustrated in FIG. 5. Underlined arerestriction linkers attached to each primer.

M3L-52 AAAGGATCCATTTAGTCT SEQUENCE ID NO:11:    BamHI M3L-31GAATTCATGTGAAAGGCGGCCGCTGA SEQUENCE ID NO:12: EcoR I

The amplified product covered the polypurine tract and the entire 3′LTR.The amplified fragment was then cloned back into pCR II, resulting inCR-M3L. The BamH I-EcoR I fragment from CR-M3L replaced the same BamHI-EcoR I fragment of MFG-NEO, generating MΔE-NEO. The Nco I-BamH I Neogene was replaced with the Nco I-EamH I CAT sequence from MFG-CAT(see:Byun, J. et al., Gene Ther., 3:780-788, 1996), resulting in MΔE-CAT.

To allow comparison of the levels of gene expression, the bacterial CATgene was used as a reporter(MFG-CAT, MΔE-CAT). CAT assays were performedby standard procedures as previously described by Byun et al'spublication(see: Byun, J. et al., Gene Ther., 3:780-788, 1996). Two daysafter transfection or transduction, cells were harvested, washed oncewith PBS, and resuspended in 0.25M Tris-HCl(pH 7.5). Total proteins wereprepared by 4 to 5 cycles of freeze-thaw followed by incubation at 65°C. for 7 min. Equivalent amounts of protein were assayed for CATactivity. The percent conversion of C-chloramphenicol to its acetylatedforms was determined by quantiating the intensity of each spot with aphosphoimager(FUJIX BAS 1000).

MFG-CAT and MΔE-CAT constructs were transfected to the packaging lineCRIP, cell-free viral supernatants were used to transduce the humanpromonocytic line U937 and T lymphoid line CEM-SS, followed by CATassay(see: FIGS. 6B, 6C and 6D). In FIG. 6B, expression of MFG were setto 1, MΔE-CAT always produced levels of CAT activity similar to MFG-CATin both transfected packaging lines and transduced target cells,suggesting that deletion of residual env sequences did not significantlyaffect gene expression.

EXAMPLE 3 Deletion of the LTR U3 Sequence

To test whether the nucleotide sequence present in the LTR is essentialfor viral function other than as a promoter and also whether the LTRcould be substituted with a heterologous promoter sequence, theinventors constructed four hybrid LTRs in which retroviral sequenceswere deleted and replaced with heterologous promoter fragments ofsimilar lengths. As a model system, the inventors isolated the fourfragments from the major immediate-early promoter(MIEP) of humancytomegalovirus (HCMV) which contains sequences interacting with variouscellular transcription factors such as NF-kB, ATF, and AP1 (see: FIG.7). Various lengths of U3 were deleted and four fragments from MIEP,called CR, CCI, CCII and CP, then added to respective sites(see: FIG.10).

The chimeric promoters containing the HCMV IE promoter elements in theMLV LTR were constructed as follows: First, four HCMV IE promoterelements were amplified by PCR using 6 primers as shown in FIG. 8, andcloned into the plasmid pCR II. To 5′ and 3′ ends of each primer,restriction sites that are naturally present in the U3 of MLV were addedas indicated. The nucleotide sequences of these primers are:

C5NH SEQUENCE ID NO:12: GCTAGCGGGACTTTCCATTGACGT  Nhe I C3KP SEQUENCE IDNO:13: GGGTACCCGGGCGACTCAGTCAATCGGAGGAGGA  Kpn I CCI-5 SEQUENCE IDNO:14: CGATCGCCGCGTTACATAAC Pvu II CCI-3 SEQUENCE ID NO:15:TCTAGAGGAAACTCCCGTAAG  Xba I CCII-5 SEQUENCE ID NO:16:TCTAGAGGTTTGACTCACGG  Xba I CCII-3 SEQUENCE ID NO:17:GAGCTCCCTACCGCCCATTT  Sac I

Secondly, the cloning vector SP65(Promega, USA) was changed to RPX68, byremoving the region between Hind III and Pvu II, leaving Hind III intactand filling-in with Xba I and Sac I sites, for the convenience offurther manipulation, as illustrated in FIG. 9. Thirdly, the plasmidRPX68-M5L, the RPX68 containing the entire 5′LTR of MLV, was constructedby amplifying the same region from pMLV(see: Shinnick, T. M. et al.,Nature 293:543-548, 1981), as shown in FIG. 11. The nucleotide ofprimers used in amplifying 5′LTR of MLV are:

HHIR SEQUENCE ID NO:18:  AAGCTTATGTGAAAGACCCCTCCTG Hind III 5LBG (SEQ IDNO:20:  AGATCTGGCGCCTAGAGAAGG  Bgl II

RPX68-M5L was subjected to four different restriction digestions. Eachrestriction site used in digestions is unique, and they all cut thesites inside the 5′LTR. Four HCMV IE promoter fragments were thenisolated from the pCRII constructs containing these fragments(pCRII-CCI,pCRII-CCII, pCRII-CR, and pCRII-CP), and then used to substitute thePvuII-XbaI, XbaI-SacI, PvuII-SacI,and NheI-KpnI fragments of the LTR,generating four plasmids (RPX68-hybrid 5′LTR). The Hind III-Bgl IIfragment of MFG-CAT containing the 5′LTR, results in four M5L-chimericCAT plasmids; M5LMCP1-CAT, M5LMCP2-CAT, M5LMCP3-CAT and M5LCP-CAT. Thechimeric promoters constructed this way are summarized in FIG. 10.

Four restriction sites(NheI, PvuII, XbaI, and SacI) shown in FIG. 10 arenaturally present in U3 and their coordinates are shown in parentheses.These sites were used to clone the four HCMV MIEP fragments. Therelative position of CAAT and TATA boxes of U33 are indicated. Thenumbers shown above the LTR are the lengths of U3 (unshaded) or HCMVMIEP(shaded) that replaced a part of U3, while those in parentheses arethe coordinates of MLV based on Shinnick et alls publication(see:Shinnick, T. M. et al., Nature 293:543-548, 1981).

MCP1 contains the 264 bp HCMV IE promoter fragments in the regionbetween ˜330 (PvuII) and −152 (XbaI) of U3. In MCP2, 117 bp ofU3(XbaI-SacI) was replaced with the 144 bp MIEP promoter. In MCP3, theU3 region from −330 (PvuII) to −36 (SacII) was substituted with the 490bp HCMV promoter. In MCP2 and MCP3, the retroviral TATA box, but not theCAAT sequence, is intact. LCP has the 422 bp HCMV promoter whichcontains full promoter activity. In this construct, the entire U3 exceptfor 30 bp present at the 5 end was deleted from the hybrid LTR,resulting in an LTR where gene expression is essentially under thecontrol of the HCMV IE promoter.

The original 3′LTR in MFG-CAT was then replaced with these hybrid LTRsas shown in FIG. 12. To insert the HCMV IE promoter fragments to the3′LTR, RPX68-M3L was first constructed by amplifying the 3′LTR from pMLVand cloning it into RPX68 as shown in FIG. 12. The oligonucleotideprimers used in this amplification are M3L-51 and M3L-31 with the latterused to construct MΔE-CAT. The nucleotide sequence of M3L-51 is:

M3L-51 SEQUENCE ID NO:21: AAAGGATCCGATTAGTCCAATTTG    BamH I

As in the case of RPX68-M5L, RPX68-M3L was subjected to 4 differentrestriction digestions and the retroviral LTR fragments were replacedwith 4 HCMV IE promoter fragments to generate RPX68-hybrid 3′LTR in thesame manner as for RPX68-hybrid 5′LTR. The 4 BamH I-EcoR I fragmentsfrom RPX68-hybrid 3′LTR were then used to substitute the BamH I-EcoR Ifragment containing 3′LTR of MFG-CAT, resulting in four M3L-chimeric CATplasmids; M3LMCP1-CAT, M3LMCP2-CAT, M3LMCP3-CAT, and M3LCP-CAT(see: FIG.13A).

The four CAT constructs containing hybrid promoters in the 3′LTR,together with the parental vector MFG-CAT, were transfected to CRIPcells and cell-free supernatants used to transduce various human celllines. The level of CAT activity was measured after either transfectionof the packaging line or transduction of various cell lines. Since allconstructs have the MLV LTR at 5′ end in transfected cells, levels ofCAT activity in transfected CRIP cells were always comparable(see: FIG.13B). The level of CAT activity was also quite similar followingtransduction of NIH3T3 and the human Tlymphoid H9 cells(see: FIGS. 13Cand 13D).

To further demonstrate that the U3 sequence deleted in the aboveexperiment is not required for viral replication or gene expression, thepresent inventors next inserted a hybrid LTR at both 5′ and 3′ ends(see:FIG. 14). In this particular examples the inventors used the hybrid LCPLTR containing the largest deletion and insertion. To construct MCC-CATcontaining the hybrid promoter at both 5′ and 3′LTR, RPX68-53LCP wasconstructed by inserting the Hind III-BgI II fragment from RPX68-M5LCPinto the Hind III-Bgl II site of RPX68-M3LCP as illustrated in FIG. 14.To provide SA and Nco I at the start codon of the env gene, pMLV wasamplified by PCR using the primer BGSA and NcoE3.

BGSA SEQUENCE ID NO:22: ATGAGATCTTATATGGGGCACC    Bgl II NcoE3 SEQUENCEID NO:23: GGATCCGGCCATGGCTGTCTAGAGGATGGTCCTCCCCCGG  BamH I   NcoI    Xba I

The amplified Bgl II-BamH I product was cloned into the Bgl II/BamH Isite of RPX68-53LCP, generating MCC. Finally, the Nco I-BamH I CATcassette was inserted into Nco I/BamH I sites of MCC, resulting inMCC-CAT(see: FIG. 14).

CRIP cells were transfected with MCC-CAT and MFG-CAT, and cell-freeviral supernatants used to transduce a variety of cell lines. Onerepresentative result is shown in Table 1. For each cell line,expression of MFG-CAT was set to 1 and that of MCC-CAT normalized to it.Because assay conditions were different among various cell lines, directcomparison between cell lines based on the above numbers should beavoided. Transductions were performed at least 3 to 5 times for eachline.

TABLE 1 Vector Cell Line MFG-CAT MCC-CAT NIH3T3 1 1.18 ± 0.21 U937 11.75 ± 0.80 H9 1 0.75 ± 0.00 CEM-SS 1 1.16 ± 0.11

As shown in Table 1, MCC always produced levels of CAT activity similarto the parental vector, following transduction of NIH3T3, U937, H9 andCEM-SS cell lines, suggesting that almost all the U3 sequence could bedeleted from both 5′ and 3′LTRs without any deleterious effects onretroviral functions.

EXAMPLE 4 Expression of the Two Genes by a Single Transcriptional Unit

The original version of MFG does not contain the selectable marker.However, expression of more than one gene would make a retroviral vectormore versatile in its application to various in vitro experiments orgene therapy trials. It has been reported that IRES elements can beinserted into MFG, allowing for expression of multiple genes from asingle polycistronic message(see: Zitvogel, L. et al., Hum. Gene Ther.,5:1493-1506, 1994; Morgan, R. A., Nucleic Acids Res., 20:1293-1229,1992; Byun et al., Gene Ther., 3:780-788, 1996). In the followingexample, the inventors tested the efficiency of gene expression in EMCVIRES-containing retroviral vectors also harboring modifications withinthe gag, env and both 5′ and 3′LTRs.

To construct retroviral vectors containing no coding sequences for gagand env, hybrid promoters in both 5′LTR and 3′LTR, CAT and NEO sequencslinked by IRES, three plasmids(pMLV, M3LMCP1-CAT, and M3LMCP3-CAT) wereamplified with the M3L-52 and M3L-31 primers used for the constructionof MΔE-CAT as shown in FIG. 15. The amplified fragments containing BamHI and EcoR I linkers at each end were used to replace the BamH I-EcoR Ifragment of MFG-CAT including the 3′LTR, resulting in MΔE-CAT,MΔEMCP1-CAT, and MΔEMCP3-CAT(Step I). The Hind III-BamH I fragments ofthe latter three plasmids containing the 5′LTR were replaced with theHind III-BamH I fragment amplified from MCC-CAT(Step II) The nucleotidesequences of primers are as follows:

SALDGAG AAGCTTGTCGACATGAGATCTTATATGGGG SEQUENCE ID NO:24: HindIII  SalICATSTOP GGATCCTTACGCCCCGCCCTGCCA SEQUENCE ID NO:25: BamH I

The small Hind III-Sal I fragments of the three intermediateplasmids(ΔGE-CAT, ΔGEMCP1-CAT, and ΔGEMCP3-CAT) were substituted by theHind III-Xho I fragment amplified from the three plasmids(MLV, M5LMCP1and M5LCP), resulting in the 4 plasmids SFG-CAT, SCP1-CAT, KCP1-CAT, andKCP3-CAT(Step III). The primers used in this step are HindIIIR and L523.Finally, the BamH I-BamH I cassette containing EMCV IRES/NEO(see: FIG.16) was inserted into the BamH I site of the 4 plasmids, generatingretroviral constructs containing hybrid promoter at both 5′ and 3′ ends(Step IV). The CAT gene was linked with NEO through EMCV-IRES. Thestructures of SCP1-CAT and KCP3-CAT constructs are summarized in FIG.17.

The BamH I-BamH I EMCV IRES-NEO cassette was constructed using pCITEcontaining EMCV IRES(Novagene, USA), and pSVTK-neo(Stratagene, USA) asshown in FIG. 16. First, the Xba I site of pCITE was converted to BamHI. Secondly, the BstX I-BamH I NEO fragment was prepared by PCR frompSVTKneo. Thirdly, the BstX I-BamH I NEO fragment was inserted to BstXI-BamH I site of pCITE-XB, whose EcoR I was subsequently converted toBamH I, resulting in pCBIN.

In summary, these retroviral vectors were constructed to containchimeric U3 at both 5′ and 3′LTRs, deletions in gag and env codingsequences, and the selectable marker NEO gene linked to CAT throughIRES(see: FIG. 17). Retroviral constructs were transfected to CRIPcells, cell-free viral supernatants used to transduce various targetcells, and the levels of CAT activity in transduced cells determined.Again, for each cell line, expression of MFG-CIN was set to 1 and thoseof MCC-CIN, SCP1-CIN, and KCP3-CIN normalized to it. Experimentalconditions were identical to others except that one of the target cellswas human foreskin fibroblasts(HFF). The result from one representativeexperiment is shown in Table 2. The novel constructs generally producedlevels of CAT activity comparable to the parental construct, suggestingthat the two gene could be efficiently expressed in the modifiedvectors.

TABLE 2 Cell Line Vector CEM-SS HFF NIH3T3 MFG-CIN 1.0 1.0 1.0 MCC-CIN1.2 1.0 1.2 SCP1-CIN 1.0 1.7 1.2 KCP3-CIN 1.7 2.0 1.7

E. coli HB101 transformed with KCP3-CIN was deposited with aninternational depository authority(IDA), the Korean Culture Center ofMicroorganisms (KCCM) on Sep. 19, 1996 as deposition No. KCCM-10110.

Use of a Different Resorter Gene

The above experiments were performed using the CAT sequence as areporter gene. To demonstrate that our observation was not restricted tospecific reporter gene, the inventors also inserted the murineGM-CSF(mGM-CSF) gene into the SCP1 retroviral vectors SCP1-mGM/CSF wasconstructed by replacing the Nco I-BamH I CAT sequence in the SCP1-CINwith the Nco I-BamH I mGM/CSF from pCRII-GM/CSF(see: Byun, J. et al.,Gene Ther., 3:780-788, 1996; FIG. 18A). The two retroviral vectorsexpressing mGM-CSF were transfected to CRIP cells(see: FIG. 18B). HFFand NIH3T3 were transduced and then selected in the presence of theantibiotic G418(see: FIGS. 18C and 18D). The same number ofdrug-resistant cells were plated on 6-cm plates and then grown foranother three days. Expression of MFG-mGM/CSF were set to 1. The levelsof murine GM-CSF production were determined by enzyme linkedimmunoadesorbent assay using the commercially available kits from R & DSystems Inc.(Minneapolis, Minn., U.S.A.; MGM00 for mGM-CSF). The newlyconstructed retroviral vector generally gave slightly higher levels ofmGM-CSF in all cell lines tested, confirming the above result based onCAT activity.

EXAMPLE 5 Role of Nco I in Gene Expression

As described, it has been speculated that the use of the Nco I site atthe env ATG in MFG is necessary to achieve high levels of proteinproduction. To test whether the initiation codon of a foreign gene hasto coincide with the ATG contained within the Nco I cloning site, thetwo EPO-expressing retroviral vectors KCP3-WNIN and KCP3-WNIN(see: FIG.19A) that differ in the presence of Nco I site, were compared. KCP3-WNINwas constructed by replacing the Nco I-BamH I CAT sequence with the NcoI-BamH I EPO fragment from pCRII-EPO(see: Byun, J. et al., Gene Ther.,3:780-788, 1996). Next, to construct a retroviral vector lacking a Nco Isite, the Nco I site of EPO was filled in by the Klenow fragment andthis filled-NcoI/Bam HI EPO gene was then inserted into thefilled-XbaI/Bam HI site of KCP3-WNIN, resulting in KCP3-WXIN. Thenucleotide sequence environmental around the Nco I site of these vectorsand the control vector MFG-WIN is shown in FIG. 19A. In summary, MFG-WINand KCP3-WNIN contain the NcoI site, while it was removed fromKCP3-WXIN.

The three constructs were transfected to CRIP cells, cell-free viralsupernatant harvested to transduce NIH3T3 cells followed by G418selection. The same number of drug-resistant cells were plated on 6-cmplates and then grown for another three days followed by ELISA(see: FIG.19B). In FIG. 19B, expression of MFG-WIN were set to 1 and those ofothers normalized to it. The levels of human EPO production weredetermined by enzyme linked immunoadsorbent assay using the commerciallyavailable kits from R & D Systems Inc. (Minneapolis, Minn., U.S.A.;U.S.A. LDEP00 for hEPO). The level of EPO produced from the constructlacking Nco I site was always comparable to the parental vector,indicating that Nco I has marginal, if any, effects on gene expressionin the cell lines tested.

EXAMPLE 6 Construction of Retroviral Vectors

Based on the above results, the present inventors constructed a seriesof retroviral vectors which accommodated the above observations, namely,retroviral vectors in which the gag and env coding sequences unnecessaryfor packaging were deleted; the U3 sequence not essential for retroviralfunctions was replaced with heterologous promoter elements; theconvenient cloning site was introduced to the truncated U3 so thatheterologous full-size promoters or promoter fragments could readily beinserted into U3; IRES was used to express more than one genes; and, theexpression site Nco I was replaced with multicloning sites.

The inventors constructed the three sets of retroviral vectors: i.e.,(1) vectors containing the chimeric U3 at both 5′ and 3′LTR(COI, CTI,COE); (2) vectors containing the original MLV U3(MOI, MTI, MOE); and,(3) vectors containing the convenient cloning site in the truncated U3(MOMT12, MOMT13).

To construct the novel, improved retroviral vectors (COI, CTI, COE) inwhich gene expression is under the control of chimeric U3, the CATsequence was removed from KCP3-CAT and the Xba I site was converted toBamH I(using pCRII-M5LCP), resulting in HCP3 as shown in FIG. 20. Thenucleotide sequence of primers used in this step are HHIR and XB5L3. Theformer primer was used for construction of RPX68-M5L.

XB5L3 SEQUENCE ID NO:26: GGATCCTCTAGAGGATGGTC BamHI  XbaI

The BamH I-Bgl II fragment containing PMDV IRES(see: Escarmis, C. etal., Virus Res., 26:113-125, 1992) was inserted into the BamH I site ofHCP3, generating COI. Subsequently, the BamH I-Sal I fragment containingEMCV IRES was inserted into the BamH I/Sal I site of COI, resulting inCTI. FMDV IRES was removed from CTI by restriction digestion with Hpa Iand Stu I, followed by ligation, generating COE. COI, CTI and COEcontain a chimeric U3, identical to that of LCP at the 5′LTR and MCP3 atthe 3′LTR. All these vectors have deletions around the gag region(likeΔ38 in FIG. 3), no env coding sequence(see: FIG. 6A), the convenientrestriction site for the gene of interest, IRES, and NEO as selectablemarkers.

E. coli HB101 transformed with CTI was deposited with an internationaldepository authority(IDA), the Korean Culture Center ofMicroorganisms(KCCM) on Sep. 8, 1997 as deposition No. KCCM-10109.

To construct another set of novel, improved retroviral vectorscontaining of the original U3 of the MLV LTR (MOI, MTI and MOE), the XbaI-BamH I fragment containing the CAT sequence was first removed fromSFG-CAT to generate HFG by amplifying the region between the 5′ end ofU3 and the naturally occuring Xba I site, just upstream from the startcodon of env, using the primers HHIR and XB5L3 (used for construction ofHCP3), fusing this Hind III-Xba I fragment with the large Hind III-Xba Ifragment of SFG-CAT as shown in FIG. 21. The BamH I-Bgl II fragmentcontaining FMDV-IRES, was then isolated from pCRII-FMDV IRES, andinserted into the BamH I site of HFG, resulting in MOI. Subsequently,the SamH I-Sal I fragment containing EMCV IRES was inserted into theBamH I/Sal I site of MOI, producing MTI. MOE was constructed by cuttingMTI with Hpa I and Stu I, and ligating these sites. Therefore, MOEcontains only the EMCV IRES.

E. coli HB101 transformed with MTI was deposited with an internationaldepository authority(IDA), the Korean Culture Center ofMicroorganisms(KCCM) on Sep. 8, 1997 as deposition No. KCCM-10108.

The present inventors also constructed the two vectors, MOMT12 andMOMT13, in which heterologous full-size promoters or promoter fragmentscan be easily inserted into the U3 region. In these vectors, the−419/−152(Nhe I/Xba I) fragment(for MOMT12) and the −419/−1 fragment(forMOMT13) were removed from U3, the restriction linkers inserted to creata multicloning site, and the truncated U3 placed at the 3′LTR of thevector. Therefore, in these retroviral vectors, heterologous fragmentscan readily be inserted to the truncated U3 and in transduced cells, thechimeric U3 is translocated to the 5′LTR to drive gene expression.

MOMT12 was constructed as illustrated in FIG. 22A. First, the twofragments were amplified from MON which is MOI containing NEO at its XhoI site by PCR using the two pairs of primers as shown in FIG. 22A andcloned into pCRII, resulting in pCRII-M1 and pCRII-M2. The two plasmidswere cut with Hind III and Nru I and the appropriate fragments ligatedwith each other as indicated in FIG. 22A, generating pCRII-MT12.pCRII-MT12 were digested with Nco I and Mlu I, and blunt-ended. MON wasdigested with Nco I and Not I, blunt-ended, and ligated with blunt-endedpCRII-MT12, resulting in MOMT12. MOMT13 was constructed in the same wayexcept that pCRII-M3 was made from MON instead of PCRII-M2(see: FIG.22B). The precise deletion end points of U3 are as indicated in FIG.22C.

The nucleotide sequences of primers used for construction of these twoplasmid are:

NEO5 SEQUENCE ID NO:27: CTCGAGATGGGATCGGCC Xha I Mhe SEQUENCE ID NO:28:TCGCGAGCGGCCGCTTGCCAAACCTACAGGTGG  Nru I  Not I Mba SEQUENCE ID NO:29:TCGCGAGTTCGAAGAGAACCATCAGATG  Nru I  Nsp V Mu53 SEQUENCE ID NO:30:ACGCGTATCGATGAAAGACCCCCGCTGACG  Mlu I  Cla I MIR SEQUENCE ID NO:31:TCGCGAGCGGCCGCCAGTCCTCCGATTG  Nru I   Not I

Test for Levels of Gene Expression and Viral Titers

To confirm that these newly constructed vectors function as expected,the inventors took the two retroviral vectors, MOI and COI, and testedthem for their levels of gene expression and viral titers.

The NEO sequence was added to the Xho I site of MOI and COI, resultingin MOIN and COIN respectively. The EPO cDNA sequence was subsequentlycloned into the BamH I site of MOIN and COIN, generating MOIN-EPO andCOIN-EPO(see: FIG. 23A). E. coli HB101 transformed with COIN-EPO wasdeposited with an international depository authority(IDA), the KoreanCulture Center of Microorganisms(KCCM) on May 15, 1997 as deposition No.KCCM-10099.

MOIN-EPO and COIN-EPO, together with the parental construct MFG-WIN,were transfected into CRIP or SING cells, and cell-free viralsupernatants were harvested to transduce NIH3T3 cells. Viral titer weredetermined 3 days post-transduction. Cells were selected in the presenceof the anibiotic G418 to be close to the actual situation.

Drug-resistant populations were obtained. Identical numbers of cellswere plated on 6-cm culture plate and the levels of EPO in the culturesupernatant compared after 3 days. The improved vector always gavehigher levels of EPO, while viral titers were comparable between thesenovel vectors and the parental MFG-based construct(see: FIG. 23B).

In addition, to confirm that these newly constructed vectors lacking theentire gag coding sequence could indeed produce viral titers comparableto MFG, the inventors transfected the amphotropic packaging line PA317with MFG-, MOI-, and COI-based retroviral vectors expressing EPO.G418-resistant PA317 populations were generated and compared for viraltiters at similar cell concentrations as previously described(see: Byunet al., Gene Ther., 3:1018-1020, 1996). As indicated in FIG. 23B, viraltiters were always comparable between the three vectors, confirming theprevious finding that the deletion of the gag coding sequence indeed hasno significant effect on viral packaging.

Preservation of Retroviral Sequences in Transduced Target Cells

The present inventors have also demonstrated, using PCR, that thenucleotide sequence in the retroviral vectors was preserved in thetransduced target cells. For this, total DNA was prepared by lysingtransduced- and selected-NIH3T3 cell line using TES(10 mM Tris-HCl(pH7.8), 1 mM EDTA, 0.7% SDS) followed by the treatment with 400 μg/mlproteinase K at 50° C. for 1 hour, phenol:chloroform extraction, andethanol precipitation. The polymerase chain reaction(PCR) was performedusing 5 μg of total genomic DNA and the following oligonucleotideprimers specific to various region of the retroviral vector as indicatedin FIG. 24.

MLba TCGCGAGTCGAAGAGAACCATCAGATG SEQUENCE ID NO:32: L228GCCTCGAGATAAGTTGCTGGCCAG SEQUENCE ID NO:33: C5NHGCTAGCGGGACTTTCCATTGACGT SEQUENCE ID NO:34: EPC5 CCATGGGGCTGCAGAATSEQUENCE ID NO:35: EPC3 GGATCCTCATTTTTGGACTGG SEQUENCE ID NO:36:

The samples were amplified through 30 cycles that included the followingparameters; denaturation at 94° for 1 min, primer annealing at 55° C.for 1 min, and primer extension at 72° C. for 1 min and 30 sec. Theamplified DNA fragments were analysed by agarose gel eletrophoresis.

If the retroviral vectors stably transfer the retroviral sequences tothe target cells, these primers would amplify 380,582, and 622 bp of the5′ region of the viral genome, EPO, and the 3′LTR from MFG-WIN orMOIN-EPO respectively, while 655, 582, and 617 bp from COIN-EPO. DNAfragments of expected lengths were present in all cells, suggesting thatthe novel vectors can stably transfer the foreign gene to target cells.

Tests for Replication-competent Virus

The producer lines containing MFG-, MOI- and COI-based retroviralvectors were shown to be free of RCR by a BAG mobilization assay andRT-PCR of the retroviral env gene.

The BAG mobilization assay was carried out as described by Pear et al'spublication(see: Pear et al., Proc. Natl. Acad. Sci., USA, 90:8392-8396,1993). Three milliliter of supernatant from producer lines was used toinfect BAG cells(see: Price et al., Proc. Natl. Acad. Sci., USA,84:156-160, 1987), and the cells were passaged 1:10 every 3 or 4 days.When passage 3 of the infected BAG cells had reached approximately 50%confluence, the medium was changed, and 24 hours later, the supernatantwas filtered with 0.45 μm pore membrane. Three milliliter of thefiltrate was used to infect NIH3T3 cells and 48 hours later, the cellswere divided into two; one was stained for β-gal, while the otherunderwent G418 selection. To determine the titer of the virus used toinfect BAG cells, 1 ml of the viral supernatant from the virus-producercells was used in parallel to infect NIH3T3 followed by G418 selection.

No X-gal stained or G418-resistant cells were found from any producerlines tested in this experiment, suggesting that at a given sensitivityof the assay, no RCR was produced from the newly constructed vectors.

Amphotropic retroviral env gene was also amplified by PCR fromrecombinant viral and transduced cellular genome. Virus-producing cellswere seeded at 5×10⁶ per 100 mm-diameter dish, and virus-containingmedium was harvested 48 hours later. Recombinant virus were harvested byultracentrifugation at 35,000 g for 2 hours in an SW50.1 after 0.45μm-pore syringe filtration. Viral pellet were resuspended in 200 ml ofTES, and 100 μg of proteinase K was added to samples and incubated 30minutes at 37° C. After a phenol-chloroform extraction, 5 U of RNasefree-DNase(Promega, U.S.A.) was added to samples and incubated 37° C.for 30 minutes. After one more phenol-chloroform extraction, RNAs wereprecipitated with ethanol and pellets were resuspended with 50 ml ofDEPC(diethylpyrocarbonate) water. Viral cDNAs were sythesized from theviral RNAs by AMV-RT(Promega, USA). Reverse transcription was initiatedfrom MLV 3′LTR-specific oligomer, MLhe and incubated with dNTPs andRNase inhibitor at 42° C. for 1 hour.

MLhe TCGCGAGCGGCCGCTGCCAAACCTACG SEQUENCE ID NO:37:

Synthesized cDNAs were used as a template of PCR. The env gene ofrecombinant viral and transduced cellular genome were amplified usingMLV-E5 and MLV-E3 primers as follows:

MLV-E5 AAGCTTATGGCGCGTTCAACGCTCTCA SEQUENCE ID NO:38: MLV-E3AAGCTTCTATGGCTCGTACTCTATAGG SEQUENCE ID NO:39:

This result was also confirmed by RT-PCR of culture supernatants fromthe producer lines using the oligonucleotide primers that can amplifythe retroviral env gene.

As clearly illustrated and demonstrated as aboves, the present inventionprovides retroviral vectors lacking coding sequences for gag and envwhich are not essential for viral functions. The vectors are safe, asthe possibility of homologous recombination between the packaging genomeand the vector is minimized, which in turn reduces the possibility ofgeneration of replication-competent retrovirus(RCR), and they allow easyinsertion of a large foreign DNA fragment. The retroviral vectors werealso designed so that the almost entire U3 can readily be substitutedwith full-size heterologous promoters or their fragments containingnucleotide sequences completely different from the original U3, wherebythe vectors can readily be converted to have their gene expressionregulated in a various and sophisticated manner. Furthermore, thevectors allow the insertion of internal ribosomal entry sites(IRES) andmulticloning sites, which renders them simultaneous expression of 2 or 3genes of interest.

39 1 17 DNA Artificial Sequence HindIIIR, single-strandedoligonucleotide 1 gcattaaagc tttgctc 17 2 24 DNA Artificial SequenceL228, single-stranded oligonucleotide 2 gcctcgagat aagttgctgg ccag 24 324 DNA Artificial Sequence L377, single-stranded oligonucleotide 3gcctcgagtc cctgggacgt ctcc 24 4 24 DNA Artificial Sequence L523,single-stranded oligonucleotide 4 gcctcgagca aaaattcaga cgga 24 5 23 DNAArtificial Sequence L739, single-stranded oligonucleotide 5 gcctcgagcagaaggtaacc caa 23 6 25 DNA Artificial Sequence R371, single-strandedoligonucleotide 6 gcctcgaggg acttcggggg gccgt 25 7 24 DNA ArtificialSequence R527, single-stranded oligonucleotide 7 gcctcgaggt ttgggaccgaagcc 24 8 24 DNA Artificial Sequence R743, single-strandedoligonucleotide 8 gcctcgagaa tggccaacct ttaa 24 9 24 DNA ArtificialSequence R1016, single-stranded oligonucleotide 9 gcctcgagcc ctcactccttctct 24 10 18 DNA Artificial Sequence ClaIL, single-strandedoligonucleotide 10 acgctcatcg ataatttc 18 11 18 DNA Artificial SequenceM3L-52, single-stranded oligonucleotide 11 aaaggatcca tttagtct 18 12 26DNA Artificial Sequence M3L-31, single-stranded oligonucleotide 12gaattcatgt gaaaggcggc cgctga 26 13 24 DNA Artificial Sequence C5NH,single-stranded oligonucleotide 13 gctagcggga ctttccattg acgt 24 14 34DNA Artificial Sequence C3KP, single-stranded oligonucleotide 14gggtacccgg gcgactcagt caatcggagg agga 34 15 20 DNA Artificial SequenceCCI-5, single-stranded oligonucleotide 15 cgatcgccgc gttacataac 20 16 21DNA Artificial Sequence CCI-3, single-stranded oligonucleotide 16tctagaggaa actcccgtaa g 21 17 20 DNA Artificial Sequence CCII-5,single-stranded oligonucleotide 17 tctagaggtt tgactcacgg 20 18 20 DNAArtificial Sequence CCII-3, single-stranded oligonucleotide 18gagctcccta ccgcccattt 20 19 25 DNA Artificial Sequence HHIR,single-stranded oligonucleotide 19 aagcttatgt gaaagacccc tcctg 25 20 21DNA Artificial Sequence 5LBG, single-stranded oligonucleotide 20agatctggcg cctagagaag g 21 21 24 DNA Artificial Sequence M3L-51,single-stranded oligonucleotide 21 aaaggatccg attagtccaa tttg 24 22 22DNA Artificial Sequence BGSA, single-stranded oligonucleotide 22atgagatctt atatggggca cc 22 23 40 DNA Artificial Sequence NcoE3,single-stranded oligonucleotide 23 ggatccggcc atggctgtct agaggatggtcctcccccgg 40 24 30 DNA Artificial Sequence SALDGAG, single-strandedoligonucleotide 24 aagcttgtcg acatgagatc ttatatgggg 30 25 24 DNAArtificial Sequence CATSTOP, single-stranded oligonucleotide 25ggatccttac gccccgccct gcca 24 26 20 DNA Artificial Sequence XB5L3,single-stranded oligonucleotide 26 ggatcctcta gaggatggtc 20 27 18 DNAArtificial Sequence NEO5, single-stranded oligonucleotide 27 ctcgagatgggatcggcc 18 28 33 DNA Artificial Sequence Mhe, single-strandedoligonucleotide 28 tcgcgagcgg ccgcttgcca aacctacagg tgg 33 29 28 DNAArtificial Sequence Mba, single-stranded oligonucleotide 29 tcgcgagttcgaagagaacc atcagatg 28 30 30 DNA Artificial Sequence Mu53,single-stranded oligonucleotide 30 acgcgtatcg atgaaagacc cccgctgacg 3031 28 DNA Artificial Sequence MIR, single-stranded oligonucleotide 31tcgcgagcgg ccgccagtcc tccgattg 28 32 28 DNA Artificial Sequence MLba,single-stranded oligonucleotide 32 tcgcgagttc gaagagaacc atcagatg 28 3324 DNA Artificial Sequence L228, single-stranded oligonucleotide 33gcctcgagat aagttgctgg ccag 24 34 24 DNA Artificial Sequence C5NH,single-stranded oligonucleotide 34 gctagcggga ctttccattg acgt 24 35 17DNA Artificial Sequence EPC5, single-stranded oligonucleotide 35ccatggggct gcagaat 17 36 21 DNA Artificial Sequence EPC3,single-stranded oligonucleotide 36 ggatcctcat ttttggactg g 21 37 28 DNAArtificial Sequence MLhe, single-stranded oligonucleotide 37 tcgcgagcggccgcttgcca aacctacg 28 38 27 DNA Artificial Sequence MLV-E5,single-stranded oligonucleotide 38 aagcttatgg cgcgttcaac gctctca 27 3927 DNA Artificial Sequence MLV-E3, single-stranded oligonucleotide 39aagcttctat ggctcgtact ctatagg 27

What is claimed is:
 1. A murine leukemia virus (MLV) based retroviralfor delivering a gene of interest to a target cell, wherein the entireGag, and Env, coding sequences are deleted and th comprises one or twointernal ribosomal entry sites.
 2. The retroviral vector of claim 1,wherein the gene of interest is located downstream from both a splicedonor site and a splice acceptor site.
 3. The retroviral vector of claim1, wherein the gene of interest is selected from the group consisting ofgenes which produces a hormone, an enzyme, a receptor and a drug ofinterest.
 4. The retroviral vector of claim 1, wherein all or part of atleast one U3 sequence of a 5′LTR and a 3′LTR obtained from a retrovirusis substituted with a major immediate-early promoter of humancytomegalovirus which is selected from the group consisting of CR, CCI,CCII and CP.
 5. The retroviral vector of claim 4, which furthercomprises a multicloning site at the truncated U3 of the 3′LTR, tofacilitate the insertion of the major immediate-early promoter of humancytomegalovirus or the fragment thereof which is selected from the groupconsisting of CR, CCI, CCII and CP thereto.
 6. The retroviral vector ofclaim 4, wherein the major immediate-early promoter of humancytomegalovirus is regulated at the transcription level by the aid of achemical or a biological molecule.
 7. The retroviral vector of claim 6,wherein the biological molecule is selected from the group consisting ofhormone, growth factor, enzyme, lymphokine and cytokine.
 8. Theretroviral vector of claim 1, wherein the internal ribosomal entry sitesare obtained from murine encephalomyocarditis virus (EMCV) or foot andmouth disease virus (FMDV).
 9. The retroviral vector of claim 1, whereineach of the two internal ribosomal entry sites are obtained from viralsources different from each other.
 10. The retroviral vector of claim 1,which further comprises a multicloning site downstream from the spliceacceptor site or upstream from the internal ribosomal entry sites, tofacilitate the insertion of a gene of interest.